Analytical element and method for theophylline determination having increased alkaline phosphatase isoenzyme

ABSTRACT

Theophylline can be determined with an analytical element and method which utilize the inhibition, by theophylline, of alkaline phosphatase activity on an appropriate substrate. The assay is carried out at a pH of 9 or less. The element comprises an absorbent carrier material, a suitable buffer and first and second zones in fluid contact. The isoenzyme of alkaline phosphatase and a suitable substrate for the isoenzyme are located in different zones of the element. The isoenzyme is present in one of the zones in an amount of at least about 100 I.U./m 2 . This level of isoenzyme reduces or eliminates bias caused by endogenous alkaline phosphatase and hemoglobin during an assay.

FIELD OF THE INVENTION

The present invention relates to clinical chemistry and to the assay ofbiological fluids for theophylline. More specifically, it relates to adry analytical element and a method for the determination oftheophylline in human biological fluids.

BACKGROUND OF THE INVENTION

Theophylline is a drug frequently administered for treatment of asthmaand pulmonary diseases. For the drug to be used successfully withoutserious side-effects, it must be frequently and carefully monitored in apatient because it has a relatively narrow therapeutic range of use,that is, 1-2 mg/dl.

Numerous techniques have been used to determine the amount oftheophylline in human serum. Most of these techniques have seriousdrawbacks. For example, known spectrophotometric methods require largesample volumes, extensive pretreatment and suffer from interferences bysimilarly structured xanthines, such as caffeine and theobromine. Knowngas chromatograpnic methods are more specific, but requirederivitization and are time consuming.

Nonisotropic immunoassay techniques are most frequently used becausethey provide rapid results and are simple to use. Although satisfactorysensitivity has been generally obtained with immunoassay techniques, ithas been found recently that they may produce highly elevated resultsdepending upon a patient's renal condition and the specificity of theantibody used in the assay. Moreover, immunoassays require the use ofgenerally costly reagents which have limited stability.

High performance liquid chromatography techniques are also known. Thesetechniques vary in specificity depending upon whether pretreatment ofthe test sample is carried out. Organic extraction steps are necessaryto improve the accuracy and specificity of the assay. Manychromatography methods are susceptible to interferences from a number ofsubstances including some common antibiotics. Other disadvantagesinclude the need for expensive instrumentation and a specializedtechnical staff to perform the assays.

It is known that theophylline can be determined by measuring itsinhibitory effect on alkaline phosphatase activity. However, whenassaying human biological fluids in this manner, it is known thatendogenous alkaline phosphatase can affect the assay and renderinaccurate results on the high side. Endogenous alkaline phosphatasemust then be destroyed or removed in some manner prior to the assay toavoid this problem.

In a literature article by B. Vinet and L. Zizian [Clin. Chem., 25: 8,pp. 1370-1372 (1979)], an assay for theophylline in human serum isdescribed in which the drug was extracted from the serum sample usingchloroform/isopropanol to separate the theophylline from an unknownquantity of endogenous alkaline phosphatase prior to the actualdetermination of theophylline. The amount of theophylline was determinedat pH 9.4 by measuring the amount of inhibition of bovine alkalinephosphatase activity which occurred due to the presence of theophylline.This assay has several serious drawbacks, however. It is limited tosolution assays. Further, it is slow and tedious due to the multipleextraction steps required to separate endogenous alkaline phosphatasefrom theophylline prior to actual determination of the drug.

A significant advance in the art is described in commonly assigned andcopending U.S. Ser. No. 692,473, filed Jan. 18, 1985 by Norton andentitled Analytical Element and Method for Determination of Theophyllineby Enzyme Inhibition. The element described and claimed therein,however, contains from about 10 to about 50 I.U./m² of the alkalinephosphatase isoenzyme needed for the assay. The preferred amountsdisclosed are from about 20 to about 40 I.U./m².

While the element described above provides a simple and rapid assay fortheophylline, it has been found that further improvement is needed toreduce the effect of endogenous alkaline phosphatase and hemoglobin inbiological test fluids on the assay. Assays carried out with thiselement are undesirably biased by the presence of these interferents.

SUMMARY OF THE INVENTION

The problems noted above are overcome with a dry analytical element forthe determination of theophylline comprising an absorbent carriermaterial, a buffer which maintains the pH at 9 or less during thedetermination and, in fluid contact, first and second zones, the firstzone containing at least about 100 I.U./m² of an isoenzyme of alkalinephosphatase which is capable of acting on a substrate for the isoenzymeat a pH of 9 or less, and the second zone containing a substrate for theisoenzyme.

A preferred embodiment of this invention is a dry analytical elementcomprising, in fluid contact, a porous spreading zone and at least oneadditional zone,

the element containing at least about 100 I.U./m² of an isoenzyme ofalkaline phosphatase which is capable of acting on a substrate for theisoenzyme at a pH of 9 or less, and a substrate for the isoenzyme,provided the phosphatase and substrate are in different zones of theelement,

the element further containing a buffer which is capable of maintainingthe pH at 9 or less during the determination, provided thatsubstantially all of the buffer is in the porous spreading zone.

Further, this invention provides a method for the determination oftheophylline comprising the steps of:

A. at a pH of 9 or less, contacting a sample of a biological fluidsuspected of containing theophylline with the dry analytical elementdescribed above, and

B. determining a detectable change resulting from the contact.

The present invention provides a simple and rapid assay for theophyllinehaving all of the advantages of the element and assay described andclaimed in copending U.S. Ser. No. 692,473, noted above. In addition,the assay of the present invention exhibits less interference fromendogenous alkaline phosphatase and hemoglobin in the biological testfluids.

Further, it was found that the present invention exhibits increased raterange and improved keeping independent of the improvements obtained withthe invention described and claimed in copending and commonly assignedU.S. Ser. No. 900,069 filed on even date herewith and entitledANALYTICAL ELEMENT AND METHOD FOR THEOPHYLLINE DETERMINATION USINGBUFFER IN SPREADING ZONE.

These improvements were achieved unexpectedly by significantlyincreasing the amount of alkaline phosphatase isoenzyme in the element.Specifically, the isoenzyme is present in an amount of at least about100 I.U./m² as opposed to the 10-50 I.U./m² used in the elementsdescribed in U.S. Ser. No. 692,473 noted above.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the determination of theophylline inbiological fluids, and especially human biological fluids. As usedherein, determination refers to either qualitative or quantitativemeasurements of the amount of theophylline in a test sample. Inparticular, this invention can be used to determine theophylline inhuman biological fluids which contain endogenous alkaline phosphatase(that is, naturally occurring enzyme) in any of its enzymatic forms (forexample, liver, intestinal, placental or bone). For example, thisinvention can be advantageously used to assay sera, whole blood, plasma,spinal fluid, sputum, bile, saliva, and other biological fluids. It isalso possible to use the invention to assay fluid preparations of tissuesuch as human skeletal muscle, kidney, placenta, heart, intestine, lungor other tissue. The preferred biological fluids used in the practice ofthis invention are human sera and whole blood.

Theophylline is determinable in the practice of this invention byinhibiting the activity of alkaline phosphatase, an enzyme which can acton a number of substrates to produce a detectable reaction product. Forexample, the following representative equation illustrates theproduction of a detectable dye by the action of alkaline phosphataseusing a typical substrate, p-nitrophenyl phosphate: ##STR1## The dye canthen be colorimetrically detected with suitable spectrophotometricdetection equipment. The amount of theophylline present in the testsample contacted with the substrate and enzyme is inversely proportionalto the amount of dye measured.

The present invention is practiced at a pH of 9 or less, and preferablyat a pH of from 7 to 9. As noted in U.S. Ser. No. 692,473, noted above,endogenous alkaline phosphatase in human fluids has reduced activity ata pH of 9 or less. Therefore, the presence of endogenous isoenzymes ofalkaline phosphatase in a test sample has less effect on an assay fortheophylline carried out at pH 9 or less. However, isoenzymes ofalkaline phosphatase which are not inactivated in an environment of pH 9or less can be used in the present invention to indicate the presence oftheophylline. Any isoenzyme from any suitable source which has thatdesired property, that is, activity measurable at a pH of 9 or less, isuseful in the practice of this invention. Particularly useful isoenzymesare those obtained from bovine sources, for example, tissues and organs(such as liver) of cattle or calves. Isoenzymes from various othersources (for example microorganisms, avian and nonhuman mammaliansources) are also useful. It is well within the skill of a worker inclinical chemistry to find isoenzymes which will be useful in thepractice of this invention.

One or more of a variety of alkaline phosphatase substrates can be usedin the practice of this invention. The substrate must be such that uponenzymatic reaction with the isoenzyme, a directly detectable changeoccurs. For example, the substrate is converted into one or moredetectable reaction products, such as a chromogen, fluorogen,radioisotopically labeled species, and other suitable detectableproducts. The detectable change measured during the assay can be theappearance or disappearance of such a detectable product, or the changeof one detectable product into another. Alternatively, the detectablechange can be brought about through a series of reactions which areinitiated by the action of the isoenzyme on the substrate. For example,the alkaline phosphatase isoenzyme can act on the substrate to releaseanother enzyme or reagent which then is used in one or more reactions toproduce a detectable product. The detectable product may be directlymeasurable, or require some physical separation or handling formeasurement.

In a preferred embodiment of this invention, the assay provides achromogen or fluorogen as a detectable product of the enzymaticreaction. Generally, the substrates which are useful in such reactionshave a phosphate group which is cleaved from the substrate moleculeduring the enzymatic reaction. Such substrates include organic mono- ordiesters of phosphoric acid or salts thereof. Examples of particularlyuseful substrates include p-nitrophenyl phosphate, phenolphthaleinmonophosphate, phenolphthalein diphosphate, thymolphthaleinmonophosphate, indoxyl phosphate, phenyl phosphate, α-naphtholphosphate, ⊖-naphthol phosphate, α-glycerol phosphate,o-methylfluorescein phosphate, o-carboxyphenyl phosphate, alkali metalsalts thereof and others known in the art, for example, in U.S. Pat. No.3,425,912 (issued Feb. 4, 1969 to Deutsch et al) and European PatentPublication No. 61,731 (published Oct. 6, 1982). Preferred substratesare p-nitrophenyl phosphate and 4-(4-nitro-2-methylsulfonylphenylazo)naphthol-1-phosphate.

The isoenzyme and substrate must be kept separated in different zones ofthe element until contacted with the liquid test sample. The amount ofisoenzyme in the element is critical to obtaining the advantages notedabove. The element must contain at least about 100 I.U./m² of theisoenzyme. In a preferred embodiment, the isoenzyme is present in acoverage of at least about 125 I.U./m². In the context of thisdisclosure, I.U. represents the International Unit for isoenzymeactivity defined as one I.U. being the amount of isoenzyme activityrequired to catalyze the conversion of 1 micromole of substrate perminute under standard pH and temperature conditions for the isoenzyme.

The amount of substrate in the element can be varied widely. Generallyit is present in an amount of from about 1 to about 5, and preferablyfrom about 2 to about 4, g/m². Other addenda (for example, surfactants,binders and buffers) are incorporated into the element in amounts whichare within the skill of an ordinary worker in clinical chemistry.

The assay is carried out at a pH of 9 or less, and preferably from about7 to about 9. Any suitable buffer or mixture of buffers can be used inthe practice of this invention as long as it is capable of maintainingthe pH during the assay at 9 or less. Particularly useful buffers arenitrogen-containing organic buffers, many of which are standard in theart [for example, see Good et al, Biochem, 5(2), 1966, pp. 467-477].Representative buffers include, but are not limited to, the groupconsisting of tris(hydroxymethyl)aminoethane.HCl, glycylglycine,N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid andN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. The first buffer ismost preferred.

The buffer can be located in any zone of the element. However, in apreferred embodiment described and claimed in copending and commonlyassigned U.S. Ser. No. 900,069, filed on even date herewith and notedabove, substantially all of the buffer is located in the spreading zone(described below) of the element of this invention. This means thatsubstantially little buffer is located in other zones of the element.More particularly, at least about 80% of the buffer is located in thespreading zone. The buffer is present therein in a suitable amount,depending upon the particular buffer, to maintain the pH of the reactionmixture at the desired pH of 9 or less. These buffering amounts caneasily be determined by one of ordinary skill in clinical chemistry, butis generally at least about about 1 g/m².

Other optional reagents can also be added to the element, if desired.For example, metal ion activators can be added to activate theisoenzymes. Such activators include divalent cations such as Mg⁺⁺, Co⁺⁺,Mn⁺⁺, Ca⁺⁺, Zn⁺⁺, Sr⁺⁺ and Fe⁺⁺, available in free or salt form (forexample, aspartate, acetate, chloride or sulfate). Alternatively, if thelevels of endogenous alkaline phsophatase in the test sample areabnormally high, inhibitors of the enzyme activity may be used. Usefulinhibitors include phenylalanine and tetramisole. Such inhibitorsadvantageously do not affect the activity of some nonhuman alkalinephosphatase isoenzymes.

In addition, one or more phosphate acceptors are preferably included inthe element to increase the rate of enzyme reaction when phosphatesubstrates are used. Useful phosphate acceptors include aminoalcohols orderivatives thereof, or aliphatic amines with the amino alcohols beingparticularly useful. Examples of such compounds are well known in theart.

Glycerol or another humectant can be added to one or more zones of theelement in order to reduce element curl.

The method of this invention is practiced with a dry analytical elementwhich comprises an absorbent carrier material, for example a thin sheetof a self-supporting absorbent or bibulous material, such as filterpaper or strips, which contains the buffer, isoenzyme and substratedescribed above. The element is divided into at least two zones and theisoenzyme and substrate are incorporated into individual zones. Suchelements are known in the art as test strips, diagnostic elements, dipsticks or diagnostic agents.

The reagents can be incorporated into a suitable absorbent carriermaterial by inhibition, impregnation, coating or other suitabletechnique. Useful absorbent materials are insoluble and maintain theirstructural integrity when exposed to water or biological fluids such aswhole blood or serum. Useful elements can be prepared from paper, porousparticulate structures, porous polymeric films, cellulose, glass fibers,woven and nonwoven fabrics (synthetic and nonsynthetic) and othersuitable materials. Useful materials and procedures for making suchelements are well known in the art.

The two zones can be separate layers, or finite areas within a singlelayer. They can be composed of the same or different materials andjoined by lamination or other standard techniques. Preferably, asdescribed in our U.S. Ser. No. 900,069, noted above, at least one of thetwo zones is a porous spreading zone (described below).

The two zones of the element can be self-supporting, that is composed ofmaterials strong enough to maintain its integrity. Preferably, the zonesare carried on a support. Such a support can be any suitabledimensionally stable, and preferably, transparent (that is, radiationtransmissive) material which transmits electromagnetic radiation of awavelength between about 200 and about 900 nm. A support of choice for aparticular element should be compatible with the intended mode ofdetection (reflection or transmission spectroscopy). Useful supportmaterials include paper, metal foils, polystyrene, polyester,polycarbonates, cellulose esters and others known in the art.

The porous spreading zone can be prepared from any suitable fibrous ornon-fibrous material or mixtures of either or both. The void volume andaverage pore size of this zone can be varied depending upon the useintended. For example, if whole blood or other liquid samples containinghigh molecular weight materials are to be assayed, the void volume andaverage pore size are generally greater than if serum or urine is to beassayed.

Useful spreading zones can be prepared using fibrous materials, eithermixed with a suitable binder material or woven into a fabric, asdescribed in U.S. Pat. No. 4,292,272 (issued Sept. 29, 1981 to Kitajimaet al). Alternatively, and preferably, the spreading zone is preparedfrom polymeric compositions (for example, blush polymers) or particulatematerials, as described in U.S. Pat. Nos. 3,992,158 (issued Nov. 16,1976 to Przybylowicz et al), 4,258,001 (issued Mar. 24, 1981 to Pierceet al), and U.S. Pat. No. 4,430,436 (issued Feb. 7, 1984 to Koyama etal) and Japanese Patent Publication No. 57(1982)-101760 (published June24, 1982). It is desirable that the spreading zone be isotropicallyporous, meaning that the porosity is the same in each direction in thezone as caused by interconnected spaces or pores between particles,fibers or polymeric strands.

Most preferably, the porous spreading zone is prepared as a blushpolymer layer as described in U.S. Pat. No. 3,992,158, noted above.

The elements have two essential zones, at least one of which can be aporous spreading zone. The other essential zone can be a reagent zone ora registration zone as those zones are known in the art. The element canhave other zones including, but not limited to, additional spreadingzones, radiation-blocking or radiation-filtering zones, subbing zones orbarrier zones. Preferably, there is a subbing zone located between thetwo essential zones. The subbing zone provides adhesion between zonesand helps to insure that the isoenzyme and substrate do not interactprior to the assay. All zones in the element are generally in fluidcontact with each other, meaning that fluids, reagents and reactionproducts (for example, color dyes) can pass or be transported betweensuperposed regions of adjacent zones. Preferably, the zones areseparately coated layers, although two or more zones can be a singlelayer. Besides the references noted above, suitable element componentsare described, for example, in U.S. Pat. Nos. 4,042,335 (issued Aug. 16,1977 to Clement), 4,132,528 (issued Jan. 2, 1979 to Eikenberry et al)and 4,144,306 (issued Mar. 13, 1979 to Figueras).

A preferred embodiment of this invention is an element comprising asupport having thereon, in order and in fluid contact, a layercontaining the isoenzyme described herein, a radiation-blocking layer, asubbing layer, and a porous spreading layer which contains a substratefor the isoenzyme and the buffer as described above. The isoenzyme layercan be a porous spreading layer also, but preferably, it is a reagent orregistration layer containing one or more hydrophilic binders (forexample, gelatin, vinyl pyrrolidone polymers or acrylamide polymers)surfactants, mordants, and other addenda. The subbing layer can compriseone or more subbing materials known to one skilled in the art, forexample, vinyl pyrrolidone polymers or acrylamide polymers. Theradiation-blocking layer generally includes one or more binders,surfactants and reflective materials which are known in the art.

Optionally, this preferred element can also include a second porousspreading layer which is the outermost layer of the element, and whichis generally contiguous to the first porous spreading layer. The secondporous spreading layer can be constructed of materials the same as ordifferent than those of the first porous spreading layer containing theisoenzyme substrate. For example, the first spreading layer containingthe buffer can comprise blush polymers according to U.S. Pat. No.3,992,158, noted above, and the second spreading layer can be composedof particulate materials as described above. This second spreading layercan also contain buffer if desired.

A variety of different elements, depending on the method of assay, canbe prepared in accordance with the present invention. Elements can beconfigured in a variety of forms, including elongated tapes of anydesired width, sheets, slides or chips.

The assay of this invention can be manual or automated. In general, inusing the dry elements, theophylline determination is made by taking theelement from a supply roll, chip packet or other source and physicallycontacting it with a sample (for example, less than 200 μl) of theliquid to be tested. Such contact can be accomplished in any suitablemanner, for example, by dipping or immersing the element into the sampleor, preferably, by spotting the element by hand or machine with a dropof the sample with a suitable dispensing means.

After sample application, the element is exposed to any conditioning,such as incubation or heating, that may be desirable to quicken orotherwise facilitate obtaining any test result.

The alkaline phosphatase present in the element then catalyzes reactionof the substrate at a rate based on the amount of alkaline phosphatasepresent which is not inhibited by theophylline in the sample. The rateof detectable change (for example, dye formation) due to formation ofthe reaction product is quantifiable using suitable apparatus forreflection or transmission spectrophotometry. Suitablespectrophotometric apparatus and procedures are known in the art. Othersuitable detection means include the use of fluorescencespectrophotometry, radiometry or enzyme labeling. The amount oftheophylline is inversely proportional to the measured reaction rate.

For example, when p-nitrophenyl phosphate is used as the substrate, theuninhibited enzymatic reaction produces p-nitrophenol which ismeasurable at 400 nm using a standard spectrophotometer. The rate of thequantifiable color change can then be directly related to the rate ofsubstrate reaction which, in turn, is indirectly related to theconcentration of theophylline in the sample.

In the following examples, which are provided to illustrate the practiceof the invention, the materials used were obtained as follows:

Beef liver alkaline phosphatase isoenzyme, p-nitrophenyl phosphate andtris(hydroxymethyl)aminomethane.HCl buffer from Sigma Chemical Co. (St.Louis, Mo.), polyurethane resin as ESTANE from B. F. Goodrich(Cleveland, Ohio), BRIJ 78 surfactant from Ruger (Irvington, N.J.),DAXAD 30S surfactant from W. R. Grace (Lexington, Mass.), lyophilizedalbumin from Miles Laboratories, (Elkhart, Ind.), TRITON X-100, X-200Eand X-405 surfactants from Rohm & Haas (Philadelphia, Pa.), and theremaining materials from Eastman Organic Chemicals (Rochester, N.Y.), orprepared using standard starting materials and procedures.

EXAMPLES 1-2 Comparative Examples of Elements

These examples are comparisons of an assay and element of the presentinvention to assays and elements similar to that described in copendingand commonly assigned in U.S. Ser. No. 692,473, noted above. It can beseen from the data presented below that the assay of this inventionexhibits reduced interference from endogenous alkaline phosphatase andhemoglobin over the Control assays.

The element of this invention was prepared having the following formatand components:

    ______________________________________                                                              Range                                                   ______________________________________                                        Spreading                                                                              Titanium dioxide   20-50 g/m.sup.2                                   Layer    Cellulose acetate  5-15 g/m.sup.2                                             Polyurethane resin 0.5-5 g/m.sup.2                                            (ESTANE)                                                                      TRITON X-405       0.1-10 g/m.sup.2                                           surfactant                                                                    p-Nitrophenyl phosphate                                                                          1-5 g/m.sup.2                                              BRIJ 78 surfactant 0.1-3 g/m.sup.2                                            Tris(hydroxymethyl)-                                                                             1-10 g/m.sup.2                                             aminomethane.HCl                                                              buffer (pH 8)                                                        Subbing  Poly(N--isopropyl- 0.1-1 g/m.sup.2                                   Layer    acrylamide                                                                    TRITON X-100       0.01-1 g/m.sup.2                                           surfactant                                                           Radiation                                                                              Gelatin (hardened) 1-15 g/m.sup.2                                    Blocking TRITON X-200E surfactant                                                                         0.01-1 g/m.sup.2                                  Layer    Titanium dioxide   20-50 g/m.sup.2                                            DAXAD 30S surfactant                                                                             0.05-0.5 g/m.sup.2                                         Tris(hydroxymethyl)-                                                                             0.1-1 g/m.sup.2                                            aminomethane.HCl                                                              buffer (pH 8)                                                                 Glycerol           0.1-2.5 g/m.sup.2                                          Piperazine-N,N' --bis-                                                                           0.1-1 g/m.sup.2                                            (2-hydroxypropane                                                             sulfonic acid)                                                       Regis-   Gelatin (hardened) 2-20 g/m.sup.2                                    tration  Alkaline phosphatase beef                                            Layer    liver isoenzyme    (See Table                                                                    I below)                                                   Tris(hydroxymethyl)-                                                                             0.1-1 g/m.sup.2                                            aminomethane.HCl                                                              buffer (pH 8)                                                                 Magnesium chloride 0.005-0.1 g/m.sup.2                                        TRITON X-100 surfactant                                                                          0.1-2 g/m.sup.2                                            Poly(styrene-co-N--vinyl-                                                                        0.5-1.5 g/m.sup.2                                          benzyl-N--benzyl-N,N--                                                        dimethylammonium                                                              chloride-co-divinyl-                                                          benzene)                                                                      Glycerol           0.1-2.5 g/m.sup.2                                          Lyophilized albumin                                                                              0.01-0.15 g/m.sup.2                               Poly(ethylene terephthalate)                                                  Support                                                                       ______________________________________                                    

Control elements were similarly prepared except that the amount ofalkaline phosphatase isoenzyme in the registration layer was less than100 I.U./m² (see Table I below).

The elements were used to determine theophylline in the followingmanner. Two test fluids were prepared, each containing 18.5 μg/ml oftheophylline. To one of these fluids was added 1000 I.U. of humanalkaline phosphatase. The theophylline concentration of each test fluidwas determined by dropping a 10 μl volume sample of the fluid onto theporous spreading layer of each element. During incubation at 37° C., therate of enzyme activity was measured by monitoring the absorbance ofresulting dye at 410 nm using a standard clinical chemistry analyzer.

The results are shown in Table I below. Bias (μg/ml) was determined bycomparing the predicted concentrations of paired human sera containingtheophylline with and without 1000 I.U. of human alkaline phosphatase.Acceptable bias is less than 3 μg/ml. It is apparent that the Controlelement exhibited unacceptable bias from the endogenous alkalinephosphatase whereas the elements of the present invention exhibitedsignificantly less bias.

                  TABLE I                                                         ______________________________________                                        Alkaline Phosphatase                                                                      Isoenzyme Concentration                                           Element     (I.U./m.sup.2)   Bias                                             ______________________________________                                        Control A    61              -5.12                                            Control B    93              -3.48                                            Example l   123              -2.62                                            Example 2   184              -2.05                                            ______________________________________                                    

It was also observed that this improvement in bias was achieved afterthe elements of this invention had been stored at 25° C. and 15%relative humidity for 1 week. The elements of this invention appear toexhibit improved keeping properties. All of the elements described abovewere evaluated after the keeping period by applying test fluidscontaining various amounts of theophylline (0.9-37.5 μg/ml) to thespreading layer and measuring the dye formed as described above. TableII below shows the results of these tests. The bias is acceptably low atall theophylline concentrations using the elements of the presentinvention. The Control elements did not exhibit acceptable bias (3 μg/mlor less) at all concentrations.

                  TABLE II                                                        ______________________________________                                                Bias (μg/ml)                                                              Theophylline Concentration (μg/ml)                                  Element  0.9          9.5       37.5                                          ______________________________________                                        Control A                                                                              0.1          -2.34     -6.27                                         Control B                                                                              -0.59        2.32      -3.16                                         Example 1                                                                              -0.66        0.58      1.79                                          Example 2                                                                              0.02         2.57      -0.16                                         ______________________________________                                    

In addition, the elements of this invention exhibited a rate rangeincrease up to 80% over the Control elements.

EXAMPLE 3 Comparison of Elements and Assays for Hemoglobin Bias

An element of this invention like Example 2 above was compared to aelement like Control A above in an assay for theophylline (20 μg/ml),and the bias due to hemoglobin (150 mg/dl) was evaluated. The elementswere tested as described in Examples 1 and 2 using a test fluidcontaining the indicated amounts of theophylline and hemoglobin. Thebias was determined by comparing the predicted concentrations of thefluid with and without hemoglobin at the same theophylline level.

Table III below shows the results of these tests. The element of thisinvention exhibited significantly less bias from the hemoglobin ascompared to the Control element. A bias of less than about 2 μg/ml isacceptable.

                  TABLE III                                                       ______________________________________                                        Element             Bias (μg/ml)                                           ______________________________________                                        Control             -2.03                                                     Example 3           -0.85                                                     ______________________________________                                    

The invention has been described in detail with particular reference topreferred embodiments thereof, but it will be understood that variationsand modifications can be effected within the spirit and scope of theinvention.

We claim:
 1. A dry analytical element for the determination oftheophylline comprising an absorbent carrier material, a buffer whichmaintains the pH at 9 or less during the determination and, in fluidcontact, first and second zones, said first zone containing at leastabout 100 I.U./m² of an isoenzyme of alkaline phosphatase which iscapable of acting on a substrate for said isoenzyme at a pH of 9 orless, and said second zone containing a substrate for said isoenzyme. 2.The element of claim 1 wherein said isoenzyme is present in an amount ofat least about 150 I.U./m².
 3. The element of claim 1 where in saidsubstrate is an organic mono- or diester of phosphoric acid.
 4. Theelement of claim 3 wherein said substrate is selected from the groupconsisting of p-nitrophenyl phosphate and 4-(4-nitro-2-methylsulfonylphenylazo)naphthol-1-phosphate.
 5. The element of claim 1 wherein saidisoenzyme is bovine liver alkaline phosphatase.
 6. A dry analyticalelement for the determination of theophylline comprising, in fluidcontact, a porous spreading zone and at least one additional zone,saidelement containing at least about 100 I.U./m² of an isoenzyme ofalkaline phosphatase which is capable of acting on a substrate for saidisoenzyme at a pH of 9 or less, and a substrate for said isoenzyme,provided said phosphatase and substrate are in different zones of saidelement, said element further containing a buffer which is capable ofmaintaining the pH at 9 or less during said determination, provided thatsubstantially all of said buffer is in said porous spreading zone. 7.The element of claim 6 wherein said isoenzyme is present in an amount ofat least about 150 I.U./m².
 8. The element of claim 6 wherein saidphosphatase substrate is in said spreading zone.
 9. An analyticalelement for the determination of theophylline comprising: a supporthaving thereon, in order and in fluid contact:a first layer containingat least about 150 I.U./m² of an isoenzyme of alkaline phosphatase whichis capable of acting on a substrate for said isoenzyme at a pH of 9 orless, a radiation-blocking layer, and a porous spreading layercontaining a substrate for said isoenzyme, said element furthercontaining a buffer which is capable of maintaining the pH at 9 or lessduring said determination, provided that substantially all of saidbuffer is in said porous spreading layer.
 10. The element of claim 9further comprising a subbing layer between said radiation-blocking andporous spreading layers.
 11. The element of claim 9 wherein said porousspreading layer is a blush polymer spreading layer.
 12. The element ofclaim 11 wherein said porous spreading layer comprises titanium dioxide.13. A method for the determination of theophylline comprising the stepsof:A. at a pH of 9 or less, contacting a sample of a biological fluidsuspected of containing theophylline with a dry analytical elementcomprising an absorbent carrier material, a buffer which maintains thepH at 9 or less during the determination and, in fluid contact, firstand second zones, said first zone containing at least about 100 I.U./m²of an isoenzyme of alkaline phosphatase which is capable of acting on asubstrate for said isoenzyme at a pH of 9 or less, and said second zonecontaining a substrate for said isoenzyme, and B. determining adetectable change resulting from said contact.
 14. The method of claim13 carried out at a pH of from about 7 to about
 9. 15. The method ofclaim 13 wherein said biological fluid is human blood serum or wholeblood.
 16. A method for the determination of theophylline comprising thesteps of:A. at a pH of 9 or less, contacting a sample of a humanbiological fluid suspected of containing theophylline with a dryanalytical element comprising: a support having thereon, in order and influid contact:a first layer containing at least about 150 I.U./m² of anisoenzyme of alkaline phosphatase which is capable of acting on asubstrate for said isoenzyme at a pH of 9 or less, a radiation-blockinglayer, and a porous spreading layer containing a substrate for saidisoenzyme, said element further containing a buffer which is capable ofmaintaining the pH at 9 or less during said determination, provided thatsubstantially all of said buffer is in said porous spreading layer, andB. determining a detectable change resulting from said contact.
 17. Themethod of claim 16 carried out at a pH of from about 7 to about
 9. 18.The method of claim 16 wherein said human biological fluid is bloodserum or whole blood.